EpiPro, a Novel, Synthetic, Activity-Regulated Promoter That Targets Hyperactive Neurons in Epilepsy for Gene Therapy Applications.

Epileptogenesis is characterized by intrinsic changes in neuronal firing, resulting in hyperactive neurons and the subsequent generation of seizure activity. These alterations are accompanied by changes in gene transcription networks, first with the activation of early-immediate genes and later with the long-term activation of genes involved in memory. Our objective was to engineer a promoter containing binding sites for activity-dependent transcription factors upregulated in chronic epilepsy (EpiPro) and validate it in multiple rodent models of epilepsy. First, we assessed the activity dependence of EpiPro: initial electrophysiology studies found that EpiPro-driven GFP expression was associated with increased firing rates when compared with unlabeled neurons, and the assessment of EpiPro-driven GFP expression revealed that GFP expression was increased ~150× after status epilepticus. Following this, we compared EpiPro-driven GFP expression in two rodent models of epilepsy, rat lithium/pilocarpine and mouse electrical kindling. In rodents with chronic epilepsy, GFP expression was increased in most neurons, but particularly in dentate granule cells, providing in vivo evidence to support the "breakdown of the dentate gate" hypothesis of limbic epileptogenesis. Finally, we assessed the time course of EpiPro activation and found that it was rapidly induced after seizures, with inactivation following over weeks, confirming EpiPro's potential utility as a gene therapy driver for epilepsy.

Drug-Inducible Gene Therapy Effectively Reduces Spontaneous Seizures in Kindled Rats but Creates Off-Target Side Effects in Inhibitory Neurons.

Over a third of patients with temporal lobe epilepsy (TLE) are not effectively treated with current anti-seizure drugs, spurring the development of gene therapies. The injection of adeno-associated viral vectors (AAV) into the brain has been shown to be a safe and viable approach. However, to date, AAV expression of therapeutic genes has not been regulated. Moreover, a common property of antiepileptic drugs is a narrow therapeutic window between seizure control and side effects. Therefore, a long-term goal is to develop drug-inducible gene therapies that can be regulated by clinically relevant drugs. In this study, a first-generation doxycycline-regulated gene therapy that delivered an engineered version of the leak potassium channel Kcnk2 (TREK-M) was injected into the hippocampus of male rats. Rats were electrically stimulated until kindled. EEG was monitored 24/7. Electrical kindling revealed an important side effect, as even low expression of TREK M in the absence of doxycycline was sufficient to cause rats to develop spontaneous recurring seizures. Treating the epileptic rats with doxycycline successfully reduced spontaneous seizures. Localization studies of infected neurons suggest seizures were caused by expression in GABAergic inhibitory neurons. In contrast, doxycycline increased the expression of TREK-M in excitatory neurons, thereby reducing seizures through net inhibition of firing. These studies demonstrate that drug-inducible gene therapies are effective in reducing spontaneous seizures and highlight the importance of testing for side effects with pro-epileptic stressors such as electrical kindling. These studies also show the importance of evaluating the location and spread of AAV-based gene therapies in preclinical studies.

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy.

It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.

Characterization of kindled VGAT-Cre mice as a new animal model of temporal lobe epilepsy.

OBJECTIVE: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism. METHODS: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology. RESULTS: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus. SIGNIFICANCE: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics.

Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1 Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Mechanisms by which a CACNA1H mutation in epilepsy patients increases seizure susceptibility.

T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca(2+) channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I-II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca(2+)-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca(2+) channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.

A potassium leak channel silences hyperactive neurons and ameliorates status epilepticus.

OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.

Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Ca(v)2.3 calcium channels.

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.

Structural determinants of the high affinity extracellular zinc binding site on Cav3.2 T-type calcium channels.

Ca(v)3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca(v)3.2 and modulation by redox agents. His(191) is found on the extracellular face of the Ca(v)3.2 channel on the IS3-S4 linker and is not conserved in other Ca(v)3 channels. Mutation of the corresponding residue in Ca(v)3.1 to histidine, Gln(172), significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca(v)3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.

Orientation of palmitoylated CaVbeta2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation.

The G(q)-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca(2+) channel (Ca(V)2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca(V)beta is coexpressed with Ca(V)2.2. When palmitoylated Ca(V)beta2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca(V)beta3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca(V)beta2a may occupy an inhibitory site on Ca(V)2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca(V)beta2a relative to Ca(V)2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca(V)beta2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the alpha interacting domain (AID) of Ca(V)2.2's I-II linker. Ca(V)betas bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca(V)beta's movements to Ca(V)2.2 gating. Although Bdel1/beta2a currents exhibited more variable enhancement by SP, Bdel2/beta2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca(V)2.2 coexpressed with Ca(V)beta3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/beta2a currents, suggesting that when palmitoylated Ca(V)beta2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca(V)beta2a's palmitoyl groups directly interact with an inhibitory site on Ca(V)2.2 to block N-current inhibition by SP.

Molecular mechanisms of lipoic acid modulation of T-type calcium channels in pain pathway.

Alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid; lipoic acid) is an endogenous compound used to treat pain disorders in humans, but its mechanisms of analgesic action are not well understood. Here, we show that lipoic acid selectively inhibited native Ca(V)3.2 T-type calcium currents (T-currents) and diminished T-channel-dependent cellular excitability in acutely isolated rat sensory neurons. Lipoic acid locally injected into peripheral receptive fields of pain-sensing sensory neurons (nociceptors) in vivo decreased sensitivity to noxious thermal and mechanical stimuli in wild-type but not Ca(V)3.2 knock-out mice. Ensuing molecular studies demonstrated that lipoic acid inhibited recombinant Ca(V)3.2 channels heterologously expressed in human embryonic kidney 293 cells by oxidating specific thiol residues on the cytoplasmic face of the channel. This study provides the first mechanistic demonstration of a nociceptive ion channel modulation that may contribute to the documented analgesic properties of lipoic acid in vivo.

Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs.

Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiological role of L-type Ca(2+) channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacology T-type Ca(2+) channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compounds that block the Ca(v)3.2 T-type channel with high affinity, focusing on two classes of compounds: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compounds were tested using a validated Ca(2+) influx assay into a cell line expressing recombinant Ca(v)3.2 channels. This study identified four clinically approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC(50) < 3 microM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concentrations, contributing to their mechanism of action.

Orientation of the calcium channel beta relative to the alpha(1)2.2 subunit is critical for its regulation of channel activity.

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.

Alternative splicing within the I-II loop controls surface expression of T-type Ca(v)3.1 calcium channels.

Molecular diversity of T-type/Ca(v)3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Ca(v)3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Delta8b variant revealed enhanced current density compared to Ca(v)3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Delta8b channels at the plasma membrane. We conclude that alternative splicing of Ca(v)3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.

I-II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels.

The intracellular loops that interlink the four transmembrane domains of Ca(2+)- and Na(+)-channels (Ca(v), Na(v)) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I-II loop) in high voltage-activated (HVA) Ca(2+) channels possesses the binding site for Ca(v)beta subunits and plays significant roles in channel function, including trafficking the alpha(1) subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I-II loop of Ca(v)1/Ca(v)2 HVA channels and Ca(v)3 LVA/T-type channels, evidence for a regulatory role of the I-II loop in T-channel function has recently emerged for Ca(v)3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Ca(v)3 channels, we have performed a structure-function analysis of the I-II loop in Ca(v)3.1 and Ca(v)3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Ca(v)3.1 and Ca(v)3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Ca(v)3 channels. In contrast to findings in Ca(v)3.2, deletion of the central region of the I-II loop in Ca(v)3.1 and Ca(v)3.3 yielded a modest increase (+30%) and a reduction (-30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Ca(v)3 function by highlighting the unique role played by the intracellular I-II loop in Ca(v)3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Ca(v)3.3.

Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels.

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels.

Recent studies have demonstrated an important role for T-type Ca2+ channels (T-channels) in controlling the excitability of peripheral pain-sensing neurons (nociceptors). However, the molecular mechanisms underlying the functions of T-channels in nociceptors are poorly understood. Here, we demonstrate that reducing agents as well as endogenous metal chelators sensitize C-type dorsal root ganglion nociceptors by chelating Zn2+ ions off specific extracellular histidine residues on Ca(v)3.2 T-channels, thus relieving tonic channel inhibition, enhancing Ca(v)3.2 currents, and lowering the threshold for nociceptor excitability in vitro and in vivo. Collectively, these findings describe a novel mechanism of nociceptor sensitization and firmly establish reducing agents, as well as Zn2+, Zn2+-chelating amino acids, and Zn2+-chelating proteins as endogenous modulators of Ca(v)3.2 and nociceptor excitability.

Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches.

T-type Ca(2+) channels encoded by voltage-gated Ca(2+) channel (Ca(v)) 3.1, 3.2, and 3.3 genes play important physiological roles and serve as therapeutic targets for neurological and cardiovascular disorders. Currently there is no selective T-channel blocker. To screen for such a blocker, we developed three stable cell lines expressing human recombinant Ca(v)3.1, 3.2, or 3.3 channels and then examined their usefulness in high throughput screens. All three cell lines displayed an increase in intracellular Ca(2+) in response to changes in extracellular Ca(2+) as detected with Ca(2+)-sensitive dyes using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA] or FlexStation [Molecular Devices]). The signal-to-noise ratio was 2-4. Co-expression of Ca(v)3.2 with a mouse leak K(+) channel, which by virtue of being open at rest hyperpolarizes the cell membrane, blocked the fluorescent signal. Co-addition of KCl to these cells induced a Ca(2+) signal that was similar to that observed in the cell line expressing Ca(v)3.2 alone. These results confirm that the detection of intracellular Ca(2+) increase in cells expressing Ca(v)3.2 alone results from Ca(2+) entry through channels that are open at the resting membrane potential of each cell line (i.e., window currents). Testing known drugs on Ca(v)3 channels showed that block could be reliably detected using the FlexStation assay, FLIPR assay, or voltage clamp recordings using the IonWorks HT system (Molecular Devices). These results support the use of the FLIPR window current assay for primary drug screening and high throughput patch recordings for secondary screening of novel T-channel blockers.

The I-II loop controls plasma membrane expression and gating of Ca(v)3.2 T-type Ca2+ channels: a paradigm for childhood absence epilepsy mutations.

Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca(v)3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca(v)3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca(v)3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca(v)3.2 channel function and expression.

Transfer of beta subunit regulation from high to low voltage-gated Ca2+ channels.

High voltage-activated Ca(2+) channel expression and gating is controlled by their beta subunits. Although the sites of interaction are known at the atomic level, how beta modulates gating remains to be determined. Using a chimeric approach, beta subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the alpha(1) interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where beta subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.